Liu et al. Malar J (2018) 17:348 https://doi.org/10.1186/s12936-018-2501-4 Journal

RESEARCH Open Access An odorant receptor from sinensis in China is sensitive to oviposition attractants Hongmei Liu1* , Luhong Liu2, Peng Cheng1, Xiaodan Huang1 and Maoqing Gong1*

Abstract Background: is an important for the spread of malaria in China. Olfactory-related behav- iours, particularly oviposition site seeking, ofer opportunities for disrupting the disease-transmission process. Results: This is the frst report of the identifcation and characterization of AsinOrco and AsinOR10 in An. sinensis. AsinOrco and AsinOR10 share 97.49% and 90.37% amino acid sequence identity, respectively, with related sequences in . A functional analysis demonstrated that AsinOrco- and AsinOR10-coexpressing HEK293 cells were highly sensitive to 3-methylindole, but showed no signifcant diferences in response to other test odorants when compared to DMSO. Conclusions: AsinOrco was characterized as a new member of the Orco ortholog subfamily. AsinOR10, which appears to be a member of the OR2-10 subfamily, is directly involved in identifcation of oviposition sites. This fnding will help to elucidate the molecular mechanisms underlying olfactory signaling in An. sinensis and provide many more molecular targets for eco-friendly pest control. Keywords: Anopheles sinensis, AsinOrco, AsinOR10, 3-Methylindole

Background malaria in some regions; even if the source of Malaria is one of the most important infectious diseases can be discovered and cleared in a timely, there is still a seriously endangering human health and safety. Te risk of local transmission and epidemic rebound. With World Health Organization (WHO) lists malaria with rapid globalization and implementation of the national AIDS and tuberculosis as the top three public health “Belt and Road” initiative, the number of people visit- problems globally. Malaria is also one of the most impor- ing areas of high malaria transmission, such as Africa tant -borne diseases in China. To respond pro- and Southeast Asia, for business, employment and tour- actively to the global action to eliminate malaria, China ism purposes has increased signifcantly. As a result, the launched the Malaria Action Plan [1] in 2010, which proportion of overseas imported cases, which reached clearly states that “by 2015, the country except for some 99.9% (3317/3321) in 2016, shows an increasing trend border areas of Yunnan and other areas have no local [2]. Such an increase poses a potential risk to relatively malaria cases”; “by 2020, the national malaria elimina- stable malaria-endemic areas. For example, a short-term tion.” Currently, most counties (districts) in China have and large-scale clustered imported outbreak occurred completed an assessment of malaria elimination. How- in Guangxi Province in 2013 [3]. In addition, malaria- ever, conditions are still favourable for the spread of nonendemic areas lack diagnostic awareness of imported malaria cases, and severe illness and death can occur. Anopheles sinensis, with a wide distribution and *Correspondence: [email protected]; [email protected] 1 Department of , Shandong Institute of Parasitic a large population, is an important vector for the Diseases, Shandong Academy of Medical Sciences, Jining 272033, spread of malaria in China. Te main strategy for the Shandong, People’s Republic of China elimination of malaria by the WHO is the timely and Full list of author information is available at the end of the article

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Malar J (2018) 17:348 Page 2 of 8

efective removal of infection sources and prevent- Identifcation of putative AsinOrco sequences ing spread among epidemic sites. Given the resistance Predicted amino acid sequences of An. sinensis of An. sinensis populations to commonly used insecti- (ASIS023681-RA) [18], Anopheles funestus (KF819859), cides, alternative control methods are crucially needed. Anopheles gambiae (AGAP002560-RA) and Culex pipi- Researchers have combined Bacillus thuringiensis var. ens quinquefasciatus (DQ231246) Orco orthologs were israelensis with oviposition attractants in “attract-and- obtained from VectorBase. Primers (Additional fle 1: kill” strategies [4] to collect more gravid females [5] Table S1) used for two-step RT-PCR were frst designed and [6] eggs than with control traps. As mosquitoes use based on these sequences using primer 5.0 to amplify their olfactory system to search for oviposition sites, partial gene sequences of AsinOrco and AsinOR10. Total research on these systems is of key importance. RNA extraction from female adult mosquitoes (3–7 days Te olfactory system of mainly includes olfac- old) and cDNA synthesis were performed using an RNe- tory receptors (ORs), odorant-binding proteins (OBPs) asy Mini Kit (QIAGEN, Hilden, Germany), the TURBO and olfactory receptor neurons (ORNs). Previous stud- DNA-free™ Kit (Ambion, Carlsbad, CA, USA) and ies have demonstrated that ORs can convert odour- TaKaRa PrimeScript™ RT-PCR Kit (Takara, Otsu, Shiga, stimulating chemical signals into electrical signals and Japan) following the manufacturers’ instructions. Gene- transmit nerve impulses to the dendrites of olfactory specifc primers (Additional fle 1: Table S1) were then neurons [7]. Accordingly, ORs are involved in mating, designed for 5′- or 3′-end rapid amplifcation of cDNA blood sucking, oviposition site searching and other ends (RACE) to amplify full-length coding sequences important life activities of mosquitoes. using a SMARTer™ RACE cDNA amplifcation kit (Clon- ORs in olfactory sensory neurons (OSNs) tech, Mountain View, CA, USA). include a coreceptor designated Orco (OR7) and con- ventional ligand-binding odorant receptors (ORXs). Identifcation of putative AsinOR10 sequences Orco genes from diferent species are highly conserved Nested RT-PCR primers (Additional fle 1: Table S1) [8, 9]. Other highly divergent ORs are conventional were designed based on the predicted amino acid odorant receptors, correlating with some olfactory- sequences of An. sinensis (ASIC007209-RA) [18], Culex mediated behavioural functions [10], and these ORs pipiens (FJ008065), (GU945397), have been associated with certain biological informa- Anopheles quadriannulatus (FJ008069), An. gambiae tion about odorants [11]. Consistently, AgamOR2, (AGAP009520-RA) and Anopheles stephensi (FJ008074) AgamOR5, AgamOR8 and AgamOR65 [12] are nar- OR10 orthologs. PCR was carried out using TaKaRa Tks rowly tuned to indole, 2,3-butanedione, 1-octen-3-ol, Gfex DNA Polymerase (Takara, Otsu, Shiga, Japan). PCR and 2-ethylphenol, respectively. In addition, some ORs amplifcation products were examined by 1.5% agarose respond strongly to specifc odorants; for example, gel electrophoresis and verifed by DNA sequencing (Inv- CquiOR10 [13] has been shown to respond strongly to itrogen, Shanghai, China). Te obtained sequences were 3-methylindole [14], an oviposition site volatile attract- compared with predicted AsinORs and AgamORs using ant, whereas AgamOR10 [12, 15] is highly sensitive to DNAMAN. 3-methylindole and indole. Indole [12, 16] is a volatile attractant component of both human sweat and ovi- Sequence analysis position sites. In the previous research, AablOR10 was Amino acid sequences of ORs were aligned using the linked to host- and oviposition-seeking behaviours, program ClustalW, and the neighbor-joining tree was prompting us to examine the odorant response pro- built using the MEGA 5.0 program [19]. Te membrane fle of AsinOR10. Tis study identifed AsinOrco and topology of the OR sequences was predicted using the AsinOR10 of An. sinensis and examined the odorant HMMTOP (version 2.0) and TMHMM (version 2.0) [20] response profle of AsinOR10. servers.

Expression of AsinORs in HEK293 cells Methods Mosquito rearing and blood feeding Te full-length coding sequences (CDSs) of AsinORs were cloned into the pME18s mammalian expression Anopheles sinensis (laboratory-susceptible strain) lar- plasmid [9] using specifc primers (Additional fle 1: vae and pupae were reared on yeast powder, and adults Table S1). Te DsRed coding sequence was amplifed were maintained on a 10% sugar solution at 25–27 °C and from pIRES2-DsRed plasmids (Clontech, Mountain View, 70–80% relative humidity with a photoperiod of 12:12 h. CA, USA) using primers containing the appropriate Tree-day-old adult females were blood-fed on a human restriction sites. AsinORs were cloned into the pME18s volunteer arm using standard protocols [17]. Liu et al. Malar J (2018) 17:348 Page 3 of 8

plasmid in-frame with the DsRed coding sequence [8]. Statistical analysis HEK293 (human embryo kidney 293) cells (purchased Statistical analyses of diferences in the cellular experi- from the Chinese Academy of Sciences) were cultured mental results were conducted with one-way ANOVA in an incubator at a constant temperature of 37 °C with followed by post hoc Tukey HSD tests (homogeneity of 5% ­CO2 and transiently transfected with AsinORs using variance: P > 0.05). ­Lipofectamine® 2000 Reagent (Invitrogen, Carlsbad, CA) [21]. Expression of ORs was confrmed by RT-PCR after Results 24 h; subcellular location analysis and western blotting Identifcation of putative AsinOR genes were performed after 48 h. Te two-step RT-PCR primers Full-length coding sequences for AsinOrco and and nested RT-PCR primers are provided in Additional AsinOR10 were successfully obtained based on bioin- fle 1: Table S1. Cells were lysed with RIPA bufer (50 mM formatics and homologous genes. AsinOrco, which is Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.25% sodium 1437 bp in length and encodes 479 amino acids, exhib- deoxycholate, 0.1% Triton X-100, 1% Nonidet P-40). Te its 96.66% sequence identity with predicted AsinOrco lysates were mixed with in SDS–PAGE bufer (62.5 mM (98.96%) and AgamOrco (90.61%). Similarly, AsinOR10, Tris, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 10% glyc- which is 1125 bp in length and encodes 375 (93.35%) erol, 0.02% bromophenol blue), heated at 95 °C for 5 min, amino acids, shares 100% and 80.59% identity with pre- separated by 10% SDS-PAGE gel electrophoresis and TM dicted AsinOR10 and AgamOR10, respectively. An align- transferred to a PVDF membrane ­(Immobilon -P, Mil- ment of AsinOrco (97.49%) and AsinOR10 (90.37%) lipore). Te blot was washed with TBST, incubated with amino acid sequences with related sequences in An. 5% skim milk for 60 min, and incubated overnight with gambiae is shown in Figs. 1, 2. In general, ORs display a an anti-RFP antibody (Abcam, Cambridge, US) raised high level of divergence [24]. An interesting phenome- in mice at a dilution ratio of 1:1000 in 1 × PBS or anti- non is that the ORs from diferent species have very high GAPDH antibody (Abcam, Cambridge, US) at 1:3000 sequence conservation. dilution at 4 °C. Te blot was then incubated with a horseradish peroxidase (HRP)-conjugated anti-mouse Sequence analysis IgG secondary antibody (1:4000) (Bethyl Laboratories, Montgomery, TX, USA) at room temperature for 90 min. To explore relationships among ORs from diferent spe- cies, phylogenetic tree analysis was carried out using similar OR sequences, mainly including AgamORs, Aae- Calcium‑imaging assay gORs and CquiORs. Te results revealed the existence of Forty-eight hours after transfection, AsinOR-express- diferent subgroups (Fig. 3). For this study, AsinOrco and ing cells were rinsed three times with HBSS. Fluo4-AM AgamOrco, AfunOrco, AalbOrco, AaegOrco, CquiOrco (Dojindo Laboratories, Tokyo, Japan) at a concentra- and CppOrco were found to be clustered together. Tis tion of 2 μM was added, and the cells were incubated for fnding indicates that AsinOrco belongs to the corecep- 30 min at 37 °C in the dark. Te cells were rinsed three tor subfamily, whereas AsinOR10, which is identifed as times with HBSS before the addition of fresh HBSS (con- a conventional odorant receptor, clusters with the OR2- taining ­Ca2+) [8] and tested using a panel of odorants, 10 subgroup. Among them, AsinOrco and AsinOR10 including indole, 1-octen-3-ol, 1-methylindole, 3-meth- display the highest identity with AgamOrco/AfunOrco ylindole, 2-methylphenol, 2,3-butanedione, 2-ethylphe- and AgamOR10/AsteOR10, respectively. Signifcantly, with the exception of OR7 orthologs, OR2 and OR10 are nol, and dimethyl sulfoxide (Sigma). All odorants (≥ 98% pure) were dissolved in DMSO and added to a fnal con- the most conserved ORs in the phylogenetic tree. Tis centration of ­10−6 M. sequence conservation suggests that OR10 may show an Fluorescence images were acquired using a laser scan- odorant-induced response profle similar to that of OR2. ning confocal microscope (Olympus, Japan). Te ­Ca2+ Tese interesting phenomena encouraged us to examine level is represented as relative fuorescence change (ΔF/ the odorant response profle of AsinOR10. Membrane topology predictions for AsinOrco and F0), where ΔF is the diference in peak fuorescence AsinOR10 revealed that these receptors belong to the caused by stimulation and F­ 0 is the baseline fuorescence [22, 23]. Baseline fuorescence was measured 100 s prior seven-transmembrane (TM) protein family with an intra- to adding the chemicals. Responses were quantifed by cellular amino-terminus (Fig. 4). Analysis of the primary amino acid sequence of AsinOrco shows that it contains the mean values of the maximal elevations (ΔF/F0) [8]. 328 Each odorant was assayed in triplicate per dish, and at a putative calmodulin (CaM)-binding site ( SAIKY- WVER336) identifed in DmelOrco (336SAIKYWVER344) least seven cells per dish were selected randomly. All 329 337 assays were performed in triplicate. and in AalbOrco ( SAIKYWVER ) [8]; in contrast, Liu et al. Malar J (2018) 17:348 Page 4 of 8

Fig. 1 Alignment of mosquito Orco amino acid sequences. Dark blue shading indicates residues conserved between AsinOrco and AgamOrco

Fig. 2 Alignment of mosquito OR10 amino acid sequences. Dark blue shading indicates residues conserved between AsinOR10 and AgamOR10

AsinOR10 does not have this putative CaM-binding site respond strongly. Terefore, HEK293 cells coexpressing or channel gate sequences. Te observed sequence con- AsinOrco and AsinOR10 were screened using a panel of servation supports our hypothesis that AsinOrco may odorants at a fnal concentration of ­10−6 M (Fig. 6) in cal- form a channel gate, as reported for DmelOrco [25, 26], cium-imaging experiments. Te strongest fuorescence and that it may form complexes involved in odour signal was elicited by 3-methylindole (skatole) (measured as the transduction [20, 25]. relative fuorescence change, ΔF/F0). Interestingly, except for 3-methylindole (F(7, 461) = 120.240, P < 0.005; Dunnett Heterologous expression of AsinOR10 in HEK293 cells T3 vs DMSO, 3-methylindole: P < 0.005; indole: P = 1.000; AsinOR transcripts were detected in HEK293 cells at 24 h 1-octen-3-ol: P = 1.000; 1-methylindole: P = 0.188; (Fig. 5a), and corresponding proteins at approximately 2-methylphenol: P = 0.320; 2,3-butanedione: P = 1.000; 78 kDa and 67 kDa (Fig. 5b) were identifed by western 2-ethylphenol: P = 1.000), the receptors showed no sig- blotting at 48 h. Te previous study [8] found that indi- nifcant diferences in their responses to other odorants vidual OR proteins respond weakly to certain test chemi- compared to dimethyl sulfoxide (DMSO). Te study cals but that HEK293 cells coexpressing Orco and OR interprets these results to indicate that AsinOR10 has Liu et al. Malar J (2018) 17:348 Page 5 of 8

Fig. 3 Phylogenetic relationships of mosquito ORs. Anopheles sinensis ORs are in red, Anopheles gambiae ORs are in black, Anopheles stephensi ORs are in pink, Anopheles funestus ORs are in light gray, Culex quinquefasciatus ORs are in green, pipiens ORs are in purple, ORs are in blue, and Aedes albopictus ORs are in yellow. AsinOrco was grouped into the coreceptor subfamily, and AsinOR10 was grouped into the OR2-10 subgroup

high sensitivity for 3-methylindole but very low sen- larger than that of conventional ORs. Membrane topol- sitivity for indole and other methylindoles, including ogy predictions show that AsinOrco and AsinOR10 1-methylindole. belong to the TM7 protein family and have an intracel- lular amino-terminus. In addition, AsinOrco has the Discussion putative CaM-binding site (328SAIKYWVER336) identi- Tis study is the frst report of the identifcation and fed in DmelOrco (336SAIKYWVER344) and in AalbOrco characterization of AsinOrco and AsinOR10. Although (329SAIKYWVER337) [8]. Tis conservation of structure ORs typically display a high level of divergence [24], may also account for functional similarity. Overall, iden- AsinOrco and AsinOR10 share 97.49% and 90.37% amino tifcation and functional validation of Orco orthologs are acid sequence identity with the coreceptor and OR2-10 hot research topics. In the previous study, AalbOrco was subfamilies, respectively. Tis study utilized the nomen- demonstrated to transmit olfactory signaling, but did clature for Orco [27] and found that AsinOrco exhib- not recognize odorants [8]. In fact, Orco forms a com- its at least 50% sequence identity with orthologs from plex with conventional odorant receptors and is essen- other insect species, and the predicted protein size is tial for odour signal transduction [20]. Indeed, silencing Liu et al. Malar J (2018) 17:348 Page 6 of 8

Fig. 4 Transmembrane regions of AsinORs predicted using HMMTOP and TMHMM. a Transmembrane regions of AsinOrco. b Transmembrane regions of AsinOR10

to mosquito ecology [12]. In previous studies, OR2-10 orthologs [12, 13, 21, 30] were found to be more likely to be highly sensitive to indole and 3-methylindole, attract- ants of oviposition sites, therefore, this study focused on the ability of AsinOR10 to perceive oviposition attract- ants. AsinOrco- and AsinOR10-coexpressing cells were exposed to seven odorants, including indole, 1-methyl- indole, 3-methylindole, 1-octen-3-ol, 2-methylphenol, 2,3-butanedione, and 2-ethylphenol. Indole [12, 16] is a volatile attractant of oviposition sites and human sweat. 3-Methylindole [14, 32, 33], also known as skatole, is a ubiquitous oviposition site volatile attractant and an egg raft pheromone; 1-methylindole is another methylindole Fig. 5 RT-PCR and western blotting. a AsinORs transcripts detected compound. 1-Octen-3-ol [33], a volatile attractant from by RT-PCR in HEK293 cells at 24 h after transfection. Lane M: large herbivores and humans, is known to attract some molecular weight marker in the 2000 bp series, Lane 1: AsinOrco, Lane anophelines [33, 34], and 2-methylphenol [30], identifed 2: AsinOR10, Lane 3: pME18s plasmid. b AsinORs proteins detected as the best ligand among phenols, elicits a strong electro- by western blotting of HEK293 cells at 48 h after transfection. Lane 1 physiological response from CquiOR2. 2,3-Butanedione recombinant AsinOrco-DsRed and AsinOR10-DsRed detected with anti–RFP antibody, Lane 2: pME18s plasmid [35] is a metabolic byproduct of human skin microfora, which excites narrowly tuned AgamOR5 [12], and 2-eth- ylphenol [36] is found in the urine of and evokes a strong electrophysiological response from AgamOR65 or mutation of Orco [8, 28, 29] damages normal odorant [12]. responses. Notably, the function of Orco is so similar that In contrast to DMSO, 3-methylindole elicits a fuo- some researchers [21] have even used Drosophila mela- rescence reaction (measured as relative fuorescence nogaster Orco as a heterodimerization partner to exam- change, ΔF/F0). Tis fnding is similar to previous ine the function of AalbORs. In this study, AsinOrco was results showing that CquiOR10 [13, 30], AalbOR10 [8] characterized as a new member of the Orco ortholog and AgamOR10 [12] orthologs respond sensitively to subfamily. Furthermore, HEK293 cells coexpressing 3-methylindole and thus further confrm the functional AsinOrco and AsinOR10 responded to odorants. conservation of OR10 orthologs. Regardless, CquiOR10 Conventional OR sequence homology has often been [13, 30], AalbOR10 [8] and AgamOR10 [12] responded associated with odorant specifcity [21, 30, 31], and the to a set of aromatic compounds, including each of narrow OR response to odorants may be highly relevant the methylindoles, 1-octen-3-ol and indole, using Liu et al. Malar J (2018) 17:348 Page 7 of 8

Authors’ contributions HML, LHL and PC provided the experimental data and wrote the paper. XDH participated the revision of the paper. MQG reviewed and edited the manu- script. All authors read and approved the fnal manuscript.

Author details 1 Department of Medical Entomology, Shandong Institute of Parasitic Diseases, Shandong Academy of Medical Sciences, Jining 272033, Shandong, People’s Republic of China. 2 Jining Center for Disease Control and Prevention, Jining 272033, Shandong, People’s Republic of China.

Acknowledgements Not applicable.

Competing interests The authors declare that they have no competing interests. Fig. 6 HEK293 cells coexpressing AsinOrco and AsinOR10 were Availability of data and materials challenged with a panel of odorant compounds. All odorants Not applicable. 6 2 were added to a fnal concentration of ­10− M. The ­Ca + level is

represented as the relative fuorescence change (ΔF/F0), where ΔF Consent for publication is the diference in peak fuorescence caused by stimulation and Not applicable. F0 is the baseline fuorescence. Responses were quantifed by the Ethics approval and consent to participate mean values of the maximal elevations (ΔF/F0). Maximal intracellular calcium concentrations occurred in HEK293 cells coexpressing Not applicable. AsinOrco and AsinOR10 (F 120.240, P < 0.005; Dunnett T3 (7, 461) = Funding vs DMSO, 3-methylindole: P < 0.005; indole: P 1.000; 1-octen-3-ol: = This work was supported by grants from the National Natural Science Founda- P 1.000; 1-methylindole: P 0.188; 2-methylphenol: P 0.320; = = = tion of China [81702034 (HML),81471985 (MQG),81672059 (MQG)], China 2,3-butanedione: P 1.000; 2-ethylphenol: P 1.000) = = Postdoctoral Science Foundation funded project [2017M622733 (HML)], medical and health science and technology development project of Shan- dong Province [2016WS0393 (HML)] and the Innovation Project of Shandong Academy of Medical Sciences. the Xenopus Oocyte System or the Drosophila mela- nogaster “empty neuron” system, whereas AsinOR10 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- showed no signifcant diferences in responses to lished maps and institutional afliations. indole, 1-octen-3-ol and 1-methylindole compared to DMSO in HEK293 cells. Tese results might be due to Received: 29 June 2018 Accepted: 1 October 2018 diferences in the intracellular epitope tags of these sys- tems, which may infuence the selectivity of the recep- tor, or this OR might not be responsive to the chemicals References tested. Despite the use of a heterogeneous expression 1. Lei ZL, Wand LY. Control situation and primary task of key parasitic system, the results indicate that AsinOR10 is directly diseases in China. Chin J Parasitol Parasit Dis. 2012;35:1–5 (in Chinese). involved in oviposition site-seeking behaviour. 2. Zhang Li FJ, Zhang SS, Jiang S, Xia ZG, Zhou SS. Malaria situation in the People’s Republic of China in 2016. Chin J Parasitol Parasit Dis. 2017;35:515–9 (in Chinese). 3. Lin Kang-Ming LJ, Yang YC, Wei SJ, Huang YM, Li JH, et al. Characteris- Conclusions tics of imported malaria epidemic in Guangxi, 2013. Modern Preventive Med. 2015;42:2439–42 (in Chinese). In summary, AsinOrco was characterized as a new 4. Barbosa RM, Regis L, Vasconcelos R, Leal WS. Culex mosquitoes member of the Orco ortholog subfamily, and AsinOR10 (Diptera: Culicidae) egg laying in traps loaded with Bacillus thur- was found to be a member of the OR2-10 subfamily. ingiensis variety israelensis and baited with skatole. J Med Entomol. 2010;47:345–8. AsinOR10 is directly involved in oviposition site iden- 5. Mboera LEG, Takken W, Mdira KY, Pickett JA. Sampling gravid Culex tifcation. Tese results will help in exploration of the quinquefasciatus (Diptera: Culicidae) in Tanzania with traps baited with molecular mechanism underlying the olfactory signal synthetic oviposition pheromone and grass infusions. J Med Entomol. 2000;37:172–6. transduction pathway in An. sinensis and provide more 6. Leal WS, Barbosa RM, Xu W, Ishida Y, Syed Z, Latte N, et al. Reverse and molecular targets for eco-friendly pest control. conventional chemical ecology approaches for the development of oviposition attractants for Culex mosquitoes. PLoS One. 2008;3:e3045. 7. Kaupp UB. Olfactory signalling in vertebrates and insects: diferences and commonalities. Nat Rev Neurosci. 2010;11:188–200. 8. Liu H, Liu T, Xie L, Wang X, Deng Y, Chen CH, et al. Functional analysis Additional fle of Orco and odorant receptors in odor recognition in Aedes albopictus. Parasit Vectors. 2016;9:363.

Additional fle 1: Table S1. List of oligonucleotide primers. Liu et al. Malar J (2018) 17:348 Page 8 of 8

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